导入TaNHX2基因提高了转基因普那菊苣的耐盐性

我国部分地区土地盐碱化的日益严重,对作物的生长和生态环境产生了显著影响,因此通过植物基因工程手段培育耐盐碱的转基因作物品种对改善作物的生存能力和生态环境,提高作物产量具有重要的意义。采用农杆菌介导法将来自小麦(Triticum aestivum Linn)的Na⁺ /H⁺逆向转运蛋白的基因(vacuolar Na⁺/H⁺ exchanger or antiporter,简称NHX、NHE或NHA),对普那菊苣(Cichorium intybus L.cv.Puna)植株进行了遗传转化。经抗生素筛选以及针对TaNHX2基因的PCR检测和Southern杂交分析,证明获得了28株转TaNHX2基因的普那菊苣植株。用不同浓度NaCl溶液对普那菊苣野生型和T₀代种子、愈伤组织和幼苗生长情况胁迫的研究,结果表明:转TaNHX2基因普那菊苣植株表现出一定的抗性,比野生型明显提高。在300 mmol/L NaCl胁迫下转基因植株种子的出芽率、外植体出愈率和分化率是野生型植株的2-4倍,而500 mmol/L NaCl浓度为野生型和转基因外植体能否生长的临界点。在此临界值下野生型外植体或不能形成愈伤组织、或幼苗不能正常生根、或已生根幼苗不能正常成长,而转基因外植体可以继续形成愈伤组织并正常生根生长。同时对500 mmol/L NaCl胁迫下野生型和转基因普那菊苣幼苗其体内丙二醛含量(MDA)、过氧化氢酶(POD)和超氧化物歧化酶(SOD)活性进行测定,结果表明 转基因植株比野生型植株的MDA含量降低了1-3倍,POD活性提高了1-3倍,SOD活性提高了2-3倍,分析发现普那菊苣的耐盐性与其体内的丙二醛含量(MDA)、过氧化氢酶(POD)和超氧化物歧化酶(SOD)活性密切相关。

Introduction of TaNHX2 gene enhanced salt tolerance of transgenic puna chicory plants

The growing severity of land salinization and alkalization in some areas of China has resulted in adverse impacts on crop production and agriculture-ecological environment. Soil salinity is one of the major limiting factors affecting crop growth, development and yield in arid and semiarid regions of China. Therefore, breeding for saline and alkaline tolerant crop varieties through genetic engineering approaches is of great significance for improving the crop survivability in saline and alkaline conditions and ecological environment, as well as for enhancing crop production. With the development of molecular biology and genetic transformation technology, numerous genes related to saline and alkaline tolerances have been found and cloned. Introduction of these genes into various plants has achieved rapid improvement of their tolerances saline and alkaline conditions. Cichorium intybus L. cv. Puna. belonging to Asteraceae family is commonly known as witloof cichory and widely distributed in northern, and central China. In this study, a wheat (Triticum aestivum Linn)vacuolar Na⁺/H⁺ exchanger gene (TaNHX2), in connection with plant salt tolerance, was introduced into puna chicory using Agrobacterium-mediated transformation approach. Puna chicory leaf segments were precultured for 2-3 days before being co-cultivated with the Agrobacterium strain, pBin438-TaNHX, which containing the target gene₂ Transformed buds were selected on the MS medium containing kanamycin (80mg/L) and Cefalexin (1000mg/L), and 28 putative transgenic lines were obtained and used for further molecular and biological assay. Analysis of the transgenic plants was performed by using PCR and Southern blot hybridization, which proved that the wheat vacuolar Na⁺/H⁺ exchanger gene was successfully integrated into puna chicory genome. Effects of NaCl concentration on growth of wild type and T₀ seed germination, callus induction and seedling growth were investigated. The results indicated that transgenic puna chicory explants tolerated certain concentrations of NaCl up to 500 mmol/L, which was much higher than that of the wild type. Under 300 mmol/L NaCl stress, the transgenic seeds germinating rate,callus induction rate and bud regeneration rate were 2-4 times higher than the wide type. NaCl concentration of 500 mmol/L was the maximum amount for the survival of wide type puna chicory plantlets, under which transgenic explants could form calli, buds, and roots, and grow normally but the wild type explants could not.. We also measured the contents of malonaldehyde (MDA), and activities of peroxidase (POD) and superoxide dismutase (SOD) in transgenic puna chicory seedlings and its wild counterpart. Under the stress of 500 mmol/L NaCl the MDA content was decreased by 1-3 times, superoxide dismutase (SOD) activity was increased by 2-3 times and peroxidase (POD) activity was increased by 1-3 times compared with those in wide type plants. The decrease of MDA content in transgenic puna chicory seedlings was negatively correlated and the increases of the enzyme activities in them were positively correlated with their tolerance to NaCl. Above all, we can make a conclusion that salt-tolerant transgenic puna chicory plants, could be obtained by introducing wheat vacuolar Na⁺/H⁺ exchanger gene into by plant engineering approaches.